Separation of very large DNA molecules by gel electrophoresis.

Very large DNA molecules were separated by electrophoresis in horizontal slab gels of dilute agarose. Conditions of electrophoresis were developed using intact DNA molecules from the bacterial viruses lambda, T4 and G. Their DNAs have molecular weights (M) of 32 million, 120 million, and 500 million, respectively. Several electrophoresis conditions were found which give sufficiently high mobilities and large differences that these DNAs are separated in a short time. Electrophoresis in 0.1% agarose at 2.5 V/cm of gel length separates T4 and lambda DNAs by 2.0 cm, and G and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilities are directly proportional to log M for M values from 10 to 500 million. The procedures used will allow rapid molecular weight determination and separation of very large DNA molecules.     

Fractionation of high molecular-weight duplex DNA molecules on dilute agarose gels

Fractionation of high molecular weight duplex DNAs was studied by electrophoresis on 0.05 to 1% agarose gels. Only the most diluted gels allowed the separation of T4 and T7 phages DNAs. The method makes use of progressive melting of the gel columns and may be applied to composite acrylamide agarose gels.     

Method of mapping DNA replication origins.

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.     

Cleavage map of bacteriophage T4 cytosine-containing DNA by sequence-specific endonucleases SalI and KpnI.

Cytosine-containing T4 DNA from endoII- endoIV- dCTPase- alc2 phage grown in a sup+ rB- mB- host is cleaved by endo R.EcoRI and endo R.HindIII to greater than 40 fragments and by endo R.SalI and endo R.KpnI to 8 and 6 fragments, respectively. The latter two fragment sets have been correlated to each other to produce a cleavage map of the genome. The sum of the molecular weights of the fragments calculated from electrophoretic mobility in agarose gels yields a genome molecular weight for cytosine-containing T4 DNA of 105 x 10(6).     

A method for the recovery of DNA from agarose gels.

We describe a quick and versatile method for the isolation of DNA from agarose gels. The DNA is electrophoresed into a trough containing hydroxyapatite, where it is bound. The hydroxyapatite is taken out and the DNA eluted with phosphate buffer. By putting the hydroxyapatite on a small column of Sephadex G50, elution and subsequent removal of phosphate can be performed in one step. The DNA recovered can be used equally well in enzymatic incubations as DNA not purified through agarose gel electrophoresis. Several applications of this technique are described.     

Electrophoresis of DNA restriction fragments in poly-N-acryloyl-tris gels

Poly-N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) gels were evaluated as a matrix for DNA electrophoresis. The resolution of DNA restriction fragments in three poly(NAT)-N,N'-methylenebisacrylamide (Bis) gels (4, 5, and 6%) was compared with the resolution in polyacrylamide (AA)-Bis gels of the same percentage. Poly(NAT) gels were found to give a substantially improved separation of DNA fragments larger than 200 bp. In contrast to poly(AA) gels, DNA fragments of up to 4 kbp were well resolved in the new matrix. By pulse-field electrophoresis the useful separation range of poly(NAT) gels was expanded to at least 23 kbp. For DNA fragments below 10 kbp, the resolution was better than that in a 0.7% agarose gel. Thus poly(NAT) gels are most suitable for the electrophoretic separation of DNA molecules whose size is out of the optimal fractionation range of poly(AA) or agarose gels.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.     

HydroLink gel electrophoresis (HLGE). III. High DNA loading capacity and recovery of dsDNA

Novel polymers have been prepared for high performance electrophoretic separations of double-stranded DNA (dsDNA). These materials are part of a family of HydroLink high performance electrophoresis polymers. A comparison of the resolving capabilities of dsDNA HydroLink gels to agarose and polyacrylamide separations has been described in an accompanying paper. In this study, we demonstrate that dsDNA HydroLink gels possess ten times the loading capacity of comparable polyacrylamide or agarose gels without compromise to resolution or biological integrity of the separated DNA. A simplified procedure for recovery of separated components is also described.     

The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.     

Electrophoresis of DNA in oriented agarose gels

Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.     

Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column

A simple and rapid method for purifying DNA from agarose gels is described. Agarose slices containing DNA are placed in a disposable plastic column and the DNA is separated from the agarose by centrifugation in a microfuge. Recoveries averaging 25% are obtained for DNA of 14 kb or less. The recovered DNA can be labeled to high specific activity, cleaved with restriction endonucleases, and ligated efficiently using standard cloning vectors.     

Purification and cloning of DNA fragments fractionated on agarose gels

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.     

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.     

Rapid alkaline transfer of low molecular weight DNA from NuSieve GTG agarose gels

The rapid alkaline transfer of high molecular weight DNA from agarose gels to nylon membranes has greatly decreased the time required for setup of Southern transfers. This technique has been used to resolve genomic DNA greater than 1000 base pairs by conventional electrophoresis on 1% agarose gels followed by alkaline transfer to nylon membrane. Now we report that this rapid alkaline method can be used for the transfer of low molecular weight DNA fragments (10 to 1000 base pairs) from NuSieve GTG agarose gels to nylon membrane.     

Single long DNA molecule analysis using fluorescence microscopy.

Single long DNA molecule (T4 DNA) in agarose gel was visualized with a fluorescence microscope. We confirmed alternating current electric fields is effective for stretching of single DNA molecule in agarose gel. This stretching phenomenon was observed with wide range of agarose gel concentration from 0.5%(W/V) to 1.5%. From this observation, the presence of agarose gel fiber is essential for this stretching phenomenon. The stretching process of several DNA molecules in gel shows discontinuity, which is never observed in polymer systems. It would be based on topological restriction from gel fibers.     

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)